The aim of this project is to successfully introduce the intact 230 kb human CFTR gene(CFTR ) and adjacent sequences into mammalian cells in order to study CFTR protein (CFTR) function and regulation of CFTR expression. The strategy is three-fold: (1) transfer human CFTR stably into non-human mammalian cells and analyse the cells for the presence of functional human CFTR. (2) transfer human CFTR stablely into human CF cells to test for correction of the CF phenotype using a YAC in which mutations have been introduced in the 3' untranslated region; (3) optimize a delivery system to transiently test for intact delivery of the human CFTR gene using the Ecoli Lac Z gene [coding for beta-galactosidase (beta-gal)] as a marker. We have investigated the function of human CFTR contained within two different yeast artificial chromosomes (YACs) introduced into Chinese hamster ovary (CHO) cells by spheroplast fusion. Both clones produced high levels of full-length, human CFTR mRNA as well as functional protein. We are also investigating the function of these two CFTR YACs in human cells from individuals with CF. A three base pair insertion introduced into the 3'-untranslated region will allow specific amplification of DNA or mRNA from the CFTR gene carried on the YAC. Finally, we are developing a transient CFTR YAC delivery using the Ecoli Lac Z gene [coding for beta-galactosidase (beta-gal)] as a marker strategy to obviate the need to generate stable cell lines.